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Key Product Details

Validated by

Knockout/Knockdown

Species Reactivity

Human, Mouse, Rat, Primate

Applications

Flow Cytometry, Immunocytochemistry/ Immunofluorescence, Immunohistochemistry, Immunohistochemistry-Paraffin, Immunoprecipitation, Knockdown Validated, Knockout Validated, Simple Western, Western Blot

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Concentration

1 mg/ml

Product Summary for EGLN1/PHD2 Antibody

Immunogen

The epitope recognized by this EGLN1/PHD2 antibody maps to a region between residues 1 and 50 of human PHD2/HIF Prolyl Hydroxylase 2 using the numbering given in entry NP_071334.1 (GeneID 54583).

Predicted Species

Orangutan (100%), Rabbit (100%), Rhesus Macaque (100%). Backed by our 100% Guarantee.

Reactivity Notes

Results for use of this EGLN1/PHD2 antibody have been mixed in Rat with success in Western blot analysis and immunofluorescence on Rat endothelial cells and negative results with PC12 cells. Mouse reactivity reported in scientific literature (PMID: 25578858). Rat reactivity reported in scientific literature (PMID: 25635047). Primate reactivity reported in scientific literature (PMID: 25974097)

Localization

Cytoplasm, nucleus

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Theoretical MW

46 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Scientific Data Images for EGLN1/PHD2 Antibody

Knockout Validated: EGLN1/PHD2 Antibody [NB100-137] - Immunoblot validation of HIF2A and PHD2 KO clones using HIF2A (#NB100-122; dilution: 1/300), PHD2 (#NB100-137; dilution: 1/500). To blot HIFs factor cells were first pre-treated for 5 h with CoCl2 300 uM before protein extraction, a condition that promotes HIF factor accumulation. Image collected and cropped by CiteAb from the following publication (http://www.nature.com/articles/s41467-018-06988-3) licensed under a CC-BY license.
Simple Western: EGLN1/PHD2 Antibody [NB100-137] - Simple Western lane view shows a specific band for PHD2/HIF Prolyl Hydroxylase 2 in 0.5 mg/mL of hypoxic HeLa cell lysate. This experiment was performed under reducing conditions using the 12-230 kDa separation system.
Western Blot: EGLN1/PHD2 Antibody [NB100-137] - Analysis of PHD2 expression in human prostate cancer cell lines. Western blot image submitted by a verified customer review.

Applications for EGLN1/PHD2 Antibody

Application
Recommended Usage

Flow Cytometry

3.0 mcg/mL

Immunocytochemistry/ Immunofluorescence

1:50

Immunohistochemistry

1:10 - 1:500

Immunohistochemistry-Paraffin

1:10 - 1:500

Simple Western

1:500

Western Blot

1:500 - 1:2500
Application Notes
This EGLN1/PHD2 antibody is useful for Flow Cytometry, Immunocytochemistry/Immunofluorescence, Western Blot, and Immunohistochemistry-paraffin embedded sections. In ICC/IF, cytoplamic and nuclear staining was observed in HeLa cells. Immunoprecipitation was reported in scientific literature.

In Simple Western only 10 - 15 uL of the recommended dilution is used per data point. Separated by Size-Wes, Sally Sue/Peggy Sue.
The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
Please Note: Optimal dilutions of this antibody should be experimentally determined.

Reviewed Applications

Read 12 reviews rated 4.8 using NB100-137 in the following applications:

Published Applications

Read 87 publications using NB100-137 in the following applications:

Formulation, Preparation, and Storage

Purification

Immunogen affinity purified

Formulation

Tris-Citrate/Phosphate (pH 7 to 8)

Preservative

0.09% Sodium Azide

Concentration

1 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at 4C. Do not freeze.

Background: EGLN1/PHD2

PHD2 (Prolyl Hydroxylase Domain-containing protein 2) belongs to the Prolyl-4-hydroxylase domain (PHD) family of proteins and is encoded by the Egl-9 Family Hypoxia Inducible Factor 1 (EGLN1) gene (1). Human EGLN1/PHD2 is a ubiquitously expressed enzyme that is 426 amino acids (aa) long with a theoretical molecular weight of ~46 kDa. Structurally PHD2 contains a nuclear export signal (NES, aa 6-20), an N-terminal MYND zinc finger domain (aa 21-58), and a C-terminal catalytic domain (aa 291-392) (2, 3). Functionally, PHD2 serves as an oxygen sensor and is responsible for post-translational modification of Hypoxia-inducible factor alpha (HIF-1alpha), a component of a transcriptional complex involved in oxygen homeostasis (1-3). During normoxia, PHD2 is responsible for oxygen-dependent hydroxylation of HIF-1alpha proline residue 402, 564, or both (3). The hydroxylation event promotes the binding of von Hippel-Lindau protein (VHL) and targets HIF1-alpha for ubiquitination and degradation (4, 5).

EGLN1/PHD2 has been implicated in several critical processes including erythropoiesis, angiogenesis, and metabolism as well as various pathologies such as cancer (2, 5, 6). Studies in mice have found that somatic deletion of PHD2 resulted in higher vascular endothelial growth factor A (VEGF-A) levels, increased blood vessel formation, and more erythropoietin (EPO), leading to severe polycythemia or erythrocytosis (high red blood cell (RBC) volume) (6). Another study revealed that specific point mutations in EGLN1/PHD2 led to elevated EPO and RBC mass associated with hemorrhages and strokes (6). Accordingly, given the known role of PHD2 in inhibition of EPO production, PHD2 inhibitors are being studied as a potential therapeutic for anemia (6). Additionally, dysregulation in EGLN1, and specifically the PHD2-VHL-HIF-1alpha pathway, has been associated with the development of pheochromocytomas (PCC) and sympathetic paragangliomas (PGL), which are rare neuroendocrine tumors (2). Besides pathological features, EGLN1/PHD2 may also be important for high altitude adaptation as two coding sequence variants in PHD2 are prevalent in the Tibetan population but is very rare in people at lower altitudes (2).

Alternate names for EGLN1/PHD2 include HIF Prolyl Hydroxylase 2, PH2, Prolyl hydroxylase domain containing protein 2, HIF2PH2, HIF-Prolyl hydroxylase 2, egl nine homolog 1, and C1orf12.

References

1. Amorim-Pires, D., Peixoto, J., & Lima, J. (2016). Hypoxia Pathway Mutations in Pheochromocytomas and Paragangliomas. Cytogenetic and genome research. https://doi.org/10.1159/000457479

2. Gardie, B., Percy, M. J., Hoogewijs, D., Chowdhury, R., Bento, C., Arsenault, P. R., Richard, S., Almeida, H., Ewing, J., Lambert, F., McMullin, M. F., Schofield, C. J., & Lee, F. S. (2014). The role of PHD2 mutations in the pathogenesis of erythrocytosis. Hypoxia (Auckland, N.Z.). https://doi.org/10.2147/HP.S54455

3. Minervini, G., Quaglia, F., & Tosatto, S. C. (2015). Insights into the proline hydroxylase (PHD) family, molecular evolution and its impact on human health. Biochimie. https://doi.org/10.1016/j.biochi.2015.07.009

4. Semenza G. L. (2007). Hypoxia-inducible factor 1 (HIF-1) pathway. Science's STKE : signal transduction knowledge environment. https://doi.org/10.1126/stke.4072007cm8

5. Chan, D. A., & Giaccia, A. J. (2010). PHD2 in tumour angiogenesis. British journal of cancer. https://doi.org/10.1038/sj.bjc.6605682

6. Meneses, A. M., & Wielockx, B. (2016). PHD2: from hypoxia regulation to disease progression. Hypoxia (Auckland, N.Z.). https://doi.org/10.2147/HP.S53576

Long Name

Egl Nine Homolog 1/Prolyl Hydroxylase Domain-containing Protein 2

Alternate Names

C1orf12, HIFPH2, HPH2, PHD2, SM20, ZMYND6

Entrez Gene IDs

54583 (Human); 112405 (Mouse); 308913 (Rat)

Gene Symbol

EGLN1

UniProt

Product Documents for EGLN1/PHD2 Antibody

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot number in the search box below.

Product Specific Notices for EGLN1/PHD2 Antibody

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

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Protocols

View specific protocols for EGLN1/PHD2 Antibody (NB100-137):

[[URL:https://www.novusbio.com/products/egln1-phd2-antibody_nb100-137]][[Capt… Prolyl Hydroxylase 2 Antibody]]
IHC-FFPE sections


I. Deparaffinization:

A. Treat slides with Xylene: 3 changes for 5 minutes each. Drain slides for 10 seconds between changes.

B. Treat slides with 100% Reagent Alcohol: 3 changes for 5 minutes each. Drain slides for 10 seconds between changes.


II. Quench Endogenous Peroxidase:


A. Place slides in peroxidase quenching solution: 15-30 minutes. To Prepare 200 ml of Quenching Solution: Add 3 ml of 30% Hydrogen Peroxide to 200 ml of Methanol.
Use within 4 hours of preparation

B. Place slides in distilled water: 2 changes for 2 minutes each.


III. Retrieve Epitopes:

A. Preheat Citrate Buffer. Place 200 ml of Citrate Buffer Working Solution into container, cover and place into steamer. Heat to 90-96 degrees Celsius.

B. Place rack of slides into hot Citrate Buffer for 20 minutes. Cover.

C. Carefully remove container with slides from steamer and cool on bench, uncovered, for 20 minutes.

D. Slowly add distilled water to further cool for 5 minutes.

E. Rinse slides with distilled water. 2 changes for 2 minutes each.


IV. Immunostaining Procedure:

A. Remove each slide from rack and circle tissue section with a hydrophobic barrier pen (e.g. Liquid Blocker-Super Pap Pen).

B. Flood slide with Wash Solution. Do not allow tissue sections to dry for the rest of the procedure.

C. Drain wash solution and apply 4 drops of Blocking Reagent to each slide and incubate for 15 minutes.

D. Drain Blocking Reagent (do not wash off the Blocking Reagent), apply 200 ul of Primary Antibody solution to each slide, and incubate for 1 hour.

E. Wash slides with Wash Solution: 3 changes for 5 minutes each.

F. Drain wash solution, apply 4 drops of Secondary antibody to each slide and incubate for 1 hour.

G. Wash slides with Wash Solution: 3 changes for 5 minutes each.

H. Drain wash solution, apply 4 drops of DAB Substrate to each slide and develop for 5-10 minutes. Check development with microscope.

I. Wash slides with Wash Solution: 3 changes for 5 minutes each.

J. Drain wash solution, apply 4 drops of Hematoxylin to each slide and stain for 1-3 minutes. Increase time if darker counterstaining is desired.

K. Wash slides with Wash Solution: 2-3 changes for 2 minutes each.

L. Drain wash solution and apply 4 drops of Bluing Solution to each slide for 1-2 minutes.

M. Rinse slides in distilled water.

N. Soak slides in 70% reagent alcohol: 3 minutes with intermittent agitation.

O. Soak slides in 95% reagent alcohol: 2 changes for 3 minutes each with intermittent agitation.

P. Soak slides in 100% reagent alcohol: 3 changes for 3 minutes each with intermittent agitation. Drain slides for 10 seconds between each change.

Q. Soak slides in Xylene: 3 changes for 3 minutes each with intermittent agitation. Drain slides for 10 seconds between each change.

R. Apply 2-3 drops of non-aqueous mounting media to each slide and mount coverslip.

S. Lay slides on a flat surface to dry prior to viewing under microscope.


NOTES:

-Use treated slides (e.g. HistoBond) to assure adherence of FFPE sections to slide.

-Prior to deparaffinization, heat slides overnight in a 60 degrees Celsius oven.

-All steps in which Xylene is used should be performed in a fume hood.

-For Epitope Retrieval, a microwave or pressure cooker may be substituted for the steamer method. Adjust times as necessary depending on conditions.

-For the initial IHC run with a new primary antibody, test tissues with and without Epitope Retrieval. In some instances, Epitope Retrieval may not be necessary.

-200 ul is the recommended maximum volume to apply to a slide for full coverage. Using more than 200 ul may allow solutions to wick off the slide and create drying artifacts. For small tissue sections less than 200 ul may be used.

-5 minutes of development with DAB Substrate should be sufficient. Do not develop for more than 10 minutes. If 5 minutes of development causes background staining, further dilution of the primary antibody may be necessary.

-Hematoxylin should produce a light nuclear counterstain so as not to obscure the DAB staining. Counterstain for 1-1.5 minutes for nuclear antigens. Counterstain for 2-3 minutes for cytoplasmic and membranous antigens. If darker counterstaining is desired increase time (up to 10 minutes).

FAQs for EGLN1/PHD2 Antibody

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  • Q: What is the probability of this antibody cross-reacting with the bovine protein. Can you send me the alignment score of the immunogen with the bovine protein?

    A: The immunogen used to generate this antibody shares 100% homology with bovine. This antibody will likely cross react with bovine, however we have not yet tested it in that species. If you would be interested in testing this species, please take a look at our Innovators Reward Program.

  • Q: I would like to use this antibody but it has not been validated in my species of interest. Is there any way I can find out if it will work?

    A: We offer risk-free testing of all of our primary antibodies. Please check out our Innovator's Reward Program and test this EGLN1-PHD2 antibody in any unvalidated species or application, without the financial risk of failure.

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